Mitotic chromosome condensation resets chromatin to safeguard transcriptional homeostasis during interphase

Abstract

Mitotic entry correlates with the condensation of the chromosomes, changes in histone modifications, exclusion of transcription factors from DNA, and the broad downregulation of transcription. However, whether mitotic condensation influences transcription in the subsequent interphase is unknown. Here, we show that preventing one chromosome to condense during mitosis causes it to fail resetting of transcription. Rather, in the following interphase, the affected chromosome contains unusually high levels of the transcription machinery, resulting in abnormally high expression levels of genes in cis, including various transcription factors. This subsequently causes the activation of inducible transcriptional programs in trans, such as the GAL genes, even in the absence of the relevant stimuli. Thus, mitotic chromosome condensation exerts stringent control on interphase gene expression to ensure the maintenance of basic cellular functions and cell identity across cell divisions. Together, our study identifies the maintenance of transcriptional homeostasis during interphase as an unexpected function of mitosis and mitotic chromosome condensation.

Keywords: cell cycle; chromatin; chromosomes; mitosis; transcription.

Fig. 1.

Failure to achieve mitotic chromosome condensation results in general upregulation of gene expression in cis. (A) The centromere excision assay. (B) RNAseq assessment of gene expression at each chromosome and at different time points after CEN4* excision. (C) RNAseq assessment of gene expression at each chromosome 180 min after CEN4* excision in CEN4* and CEN4* H3S10D cells.

Fig. 2.

Centromere loss triggers chromatin relaxation in cis. H4K12ac (A) and H4K16ac (C) occurrence at each chromosome and at different time points after CEN4* excision. Metagene analysis of the top 20% ChIP signals showing increasing presence of H4K12ac (B) and H4K16ac (D) at chromosome IV genes and decreasing presence at genes on other chromosomes after centromere excision (see SI Appendix, Fig. S9 for metagenes of 100% of ChIP signals). (E) ATACseq experiments showing chromatin accessibility at time points 0 min (Upper) and 180 min (Lower) at each chromosome after CEN4* excision in CEN4* and in CEN4 cells. Each chromosome is represented using a dedicated color. y axis: chromosome coordinates. Each dot corresponds to bin of 10 Kb of chromosomal DNA.

Fig. 3.

Lack of mitotic condensation triggers spontaneous transcriptional initiation. Assessment of H3K4me3 (A), Pol II (C), Pol II CTD-S5p (E), and Pol II CTD-S2p (G) occurrence using ChIPseq at each chromosome and at different time points after CEN4* excision. Metagene analysis of the top 20% ChIP signals shows increasing presence of H3K4me3 (B), Pol II (D), Pol II CTD-S5p (F), and Pol II CTD-S2p (H) at chromosome IV genes and a decrease at other chromosomes after centromere excision.

Fig. 4.

Failure to condense chromatin at mitotic entry results in general upregulation of gene expression during interphase. (A) Layout of CEN4* excision in S-phase arrest and release experiments. (B) RT-qPCR analysis of the expression of five selected genes from chromosome IV at different time points before and after S-phase arrest and release. *P values < 0.05; **P values < 0.01. (C) Analysis of the cell cycle using flow cytometry performed with the cells indicated in B.

Fig. 5.

Centromere excision has no effect on gene expression in cells arrested in G1 and M phases. (A) Layout of CEN4* excision in G1-phase arrest experiments (Left). RT-qPCR analysis of the expression of six selected genes from chromosome IV at different time points before and after G1-phase arrest (Right). *P values < 0.05. (B) Analysis of the cell cycle using flow cytometry performed with the cells indicated in A. (C) Layout of CEN4* excision in M-phase arrest experiments (Left). RT-qPCR analysis of the expression of six selected genes from chromosome IV at different time points before and after M-phase arrest (Right). *P values < 0.05. (D) Analysis of the cell cycle using flow cytometry performed with the cells indicated in C.